The SARS human coronavirus (SARS HuCoV) is a newly identified human coronavirus that causes Severe Acute Respiratory Syndrome (SARS), is transmitted person-to-person, and recently spread rapidly in a worldwide epidemic. The goal of this exploratory application is to begin defining the immunological mechanisms underlying SARS. The clinical course, viral kinetics, and histopathology of SARS suggest that lung pathology and respiratory compromise may be immune-mediated. Delineation of the immune responses to SARS HuCoV infection will contribute to understanding disease pathogenesis, identify components of protective immunity, and assist rational development of therapeutic and vaccine strategies. The first aim of this project will be to characterize the viral permissiveness and effector functions of key pulmonary innate immune cells to SARS HuCoV infection. This aim will use flow cytometry, virus plaquing, quantitative RT-PCR, ELISAs, mixed leukocyte reactions, and DNA microarrays, to measure virus infection and specific innate immune response effector functions. These exploratory studies will lay the foundation for future studies designed to reconstruct the viral factors and cellular mechanisms responsible for protective or pathogenic immunity. Also, immunohistochemical staining and laser capture microdissection + RT-PCR techniques will be used to identify cellular architecture and cytokine/chemokine expression patterns in autopsy lung tissue from fatal SARS cases. Study of the in vivo immune responses at the site of disease will provide valuable information regarding the pathophysiological relevance of in vitro experimental findings. The second aim will be to begin mapping antigen specificities and epitope hierarchies of SARS HuCoV-specific memory T-lymphocytes obtained from SARS survivors. CD4+ and CD8+ T-cell epitopes from SARS HuCoV structural proteins will be identified in an unbiased manner from early convalescent PBMC using overlapping synthetic peptides and ELISPOT assays. Changes in the frequency and hierarchy of antigen-specific SARS HuCoV memory T-lymphocytes will be assessed over time by ELISPOT. Elucidating the fine specificity of memory T-cells to SARS HuCoV structural proteins will lay the foundation for future studies examining antigen-presenting cell and antigenic peptide influences on SARS HuCoV-directed T-cell effector functions. Such studies will assess potential correlates of protective adaptive immunity to SARS HuCoV infection.